Project Summary The matrix metallo proteases (MMPs) are a large family of enzymes that have been found to regulate many aspects of both normal cellular biology and pathogenesis of human diseases. An overall lack of understanding of the roles of specific members of this protease family in both normal cellular processes and during specific aspects of disease progression has hindered efforts to target these proteases for therapeutic purposes. One of the confounding issues that continue to limited efforts to better define MMP function is the overall large size of the family and a lack of highly selective inhibitors for temporally controlled inhibition of specific MMP family members. Furthermore, efforts to genetically knock-out specific proteases are limited by the essential nature of many of these proteases and the potential for compensation by related family members. Thus, non-traditional methods to both selectively inhibit and image the dynamics of localization of individual MMP proteases will be required to begin to understand the specific functional roles of each MMP in both normal physiology and also during pathogenesis of human diseases such as cancer. This proposal outlines our plans to develop a novel strategy to develop small molecules that can be used to both selectively inhibit and also dynamically image individual MMP proteases in the context of a complex biological system. This method is based on the engineering of specific MMP targets to contain a reactive cysteine residue near the active site that can be used for direct covalent modification by a probe containing a reactive electrophile. Inhibitors that bind to the metal in the active site and have the ability to covalently link to the engineered cysteine residue can then be used to selectively target the cysteine variant MMPs. This allows selective covalent inhibition and labeling of a single MMP target. We describe our plans to implement this strategy into a 3D cell culture model to study the functional roles of MMP-14 and MMP-15 in both normal cellular morphogenesis and also during initiation of oncogene-derived progression to a tumor-like phenotype.